诱导多能干细胞来源的间充质干细胞的生物学特性

Biological Properties of Mesenchymal Stem Cells Derived from Induced Pluripotent Stem Cells

目的:利用外周血单个核细胞获得诱导多能干细胞(iPSC)并进一步诱导分化为间充质干细胞(MSC),观察iPSC来源MSC的生物学特性,并与其他来源MSC的相关生物学特性进行对比。方法:获取外周血单个核细胞并使用仙台病毒载体对其进行重编程因子的转导,鉴定其细胞形态和多能性因子的免疫荧光表达水平。对1株完成所有鉴定的iPSC进行MSC的诱导分化,并利用免疫组织化学和流式细胞术对其细胞形态、免疫表型进行鉴定。使用成脂、成骨培养液进行成脂成骨的诱导分化,以鉴定其分化能力。利用PCR鉴定其转录因子的表达,以系统阐明iPSC诱导分化所得的MSC的生物学特性。结果:外周血单个核细胞经过携带重编程因子的仙台病毒转染后,命运发生转归。通过启动干性基因表达,成功地获得iPSC克隆,经扩增、纯化,最后获得了可稳定增殖的iPSC。免疫荧光定位证明,其多能性因子分别在胞膜和胞内得到表达。经鉴定,利用iPSC诱导获得了间充质干细胞,其形态与其它来源的MSC一致,免疫表型检测也相符。iPSC-MSC的成脂成骨诱导分化与UC-MSC结果一致,表明iPSC-MSC具有分化(成脂成骨)能力,也表明了其具备MSC的多项基本特征。RT-PCR检测显示,iPSC-MSC高表达PDL1,低表达A20;其STAT3表达水平与BM-MSC相当,但高于UC-MSC;而HIF1α的表达水平与UC-MSC相当,但低于BM-MSC。结论:外周血单个核细胞在携带重编程因子的仙台病毒载体的转导后,成功地启动了细胞的干性表达,获得了具有多能干性的iPSC。iPSC可成功诱导分化为MSC,且分化后的MSC具备标准的MSC形态、免疫表型和分化能力。iPSC来源的MSC与脐带MSC相比,高表达免疫抑制分子PDL1并低表达A20等转录因子,提示iPSC来源细胞在细胞增殖和免疫调节等方面具有不同的生物学特性。

Objective:To obtain induced pluripatent stem cells ( iPSC) from peripheral blood mononucleated cells and further induce differentiation into mesenchymal stem cells ( MSC),and to compare the biological characteristics of iPSC-derived MSC and other-derived MSC.Methods:Peripheral blood mononucleated cells were obtained and transduced with reprogramming factors by sendai virus vector.Induced differentiation of MSC was performed in 1 strain of iPSC that completed all identification,and their cell morphology and immunophenotype were identified by immunohistochemistry and flow cytometry.Adipogenic and osteogenic media were used to induce the adipogenic and osteogenic differentiation.The expression of immune-related transcription factors was identified by PCR to systematically elucidate the biological characteristics of iPSC-induced MSC.Results:After transfection with sendai virus with reprogramming factor,the fate of peripheral blood mononucleated cells was reversed,initiating the expression of stem cell characteristics,the iPSC was successfully cloned,amplified,and purified,and finally the stable proliferation of iPSC was obtained.Mesenchymal stem cells derived from iPSC,had morphology consistent with other-derived MSC, and the immunophenotypes met the standard.iPSC-MSC possessed the ability of lipogenic and osteogenic differentiation. RT-PCR showed that iPSC-MSC was high expression to PDL1,and low expression to A20;besides,the expression level of STAT3 was equal to BM-MSC;and also as to the expression level of HIF1α and UC-MSC,which was lower than BM-MSC.Conclusion:Peripheral blood mononucleated cells successfully initiated the expression of stem cell cha- racteristics after the transduction of sendai virus vector with reprogramming factors,and obtained multi-competent iPSC. iPSC can successfully be induced to the differentiation of MSC,and the iPSC-MSC have standard cell morphology, immunophenotype and differentiation ability.High expression of PDL1 and low expression of A20 in iPSC-MSC suggest that iPSC-derived cells have different biological characteristics in cell proliferation and immune regulation.